This proposal focuses on the development of a sample-sparing and multiplexable diagnostic for detecting and monitoring the immune marker IgE against food allergen components using Isotype- Specific Agglutination-PCR (ISAP) technology. Early identification of marking IgE can prevent severe allergic reactions by enabling allergen avoidance and linking patients to targeted immunotherapies to induce allergen de-sensitization. Current food allergy IgE diagnostics mostly use whole food extract as antigens, which leads to low assay specificity due to contamination from cross-reactive proteins. Up to 77% patients positive for peanut extract IgE are not at risk for severe allergic reactions. In contrast, diagnostics that use individually purified peanut components can identify allergy risks with significantly improved accuracy. However, these component- resolved IgE diagnostics require large sample volumes and have high assay costs that adversely restrict their adoption. To address these limitations, the proposed ISAP-based allergy diagnostic uses a homogenous ligation- based DNA barcoding assay to sensitively quantify multiple allergen IgE types in a 1?L volume sample. The sample-sparing nature of the ISAP assay permits pediatric-friendly near-painless blood collection and also preserves precious samples for additional immunological testing such as genomic and cellular analysis. Notably, since this assay uses inexpensive reagents and widely-available qPCR instruments for readout, it answers the call for a low cost, yet easily-adoptable assay for both clinical and research environments. Specific Aim I seeks to synthesize and characterize ISAP reagents for a peanut allergy panel and validate them with Co-Investigators at the Stanford University Sean Parker Center for Allergy & Asthma Research using samples collected from their peanut allergy immunotherapy trial (POISED). First, we will synthesize ISAP reagents and optimize them for cross-reactivity in detecting sIgE using positive control samples. Next, we will assess potential technical pitfalls including reproducibility of the conjugation protocol, stability of key reagents and potential interference from hemolytic and lipemic samples. Finally, we will perform a head-to-head comparison of ISAP-based assays with the current standard - ImmunoCAP. The results from this aim will provide the basis for an ISAP-based allergy test that can significantly improve pediatric and adult patient compliance and disease management. Specific Aim 2 expands upon the peanut allergy panel developed in the previous aim to create a 13-plex food allergy test that covers over 80% of major food allergens. As before, we will synthesize ISAP reagents for each component and validate them using pre-characterized serum samples. A second head-to-head comparison will be performed between ISAP and ImmunoCAP using samples derived from the multi-food allergy trial (POISED and MAP-X). In an era of rising prevalence of food allergy, the study can establish ISAP as a sample-sparing assay to better serve millions of pediatric and adult patients, while providing proof-of-principle for ISAP use for the detection of a wide array of other allergens, including environmental, drug and those implicated in asthma and dermatitis.